[The ratio of tuberculosis-specific antigen to phytohemagglutinin in T-SPOT assay in the diagnosis of active tuberculosis].

Objective: The aim of this study was to determine the performance of the ratio of tuberculosis-specific antigen (TBAg) to phytohemagglutinin (PHA) (TBAg/PHA ratio) in T-SPOT assay in the diagnosis of active tuberculosis (ATB). Methods: Between January 2014 and January 2017, 378 Mycobacterium tuberculosis (MTB) culture positive patients (268 cases of pulmonary tuberculosis, 110 extra-pulmonary tuberculosis) and 824 healthy individuals were recruited from Tongji Hospital, Tongji Medical College, Huazhong University of Science and Technology. T-SPOT assay was performed and TBAg/PHA ratio was calculated in all the participants. To validate the study, another group of 223 MTB culture positive TB patients with positive T-SPOT results were recruited from Guangzhou Chest Hospital between January 2017 and December 2017. This was a retrospective case-control study and differences between groups were analyzed using the Mann-Whitney U-test. Results: Of the 378 culture positive ATB patients, 344 patients had positive T-SPOT results. Of the 824 healthy individuals, 204 individuals had positive T-SPOT results. Using healthy individuals as the control group, the sensitivity and specificity of T-SPOT assay in the diagnosis of ATB were 91.0% (344/378) and 75.2% (620/824). Directly using T-SPOT results had a limited accuracy in distinguishing ATB from latent tuberculosis infection (LTBI). The area under the receiver operating characteristic (ROC) curve was between 0.7 and 0.8. However, a further calculation of the TBAg/PHA ratio showed a better performance than TBAg in distinguishing these two conditions, and the area under the ROC curve was 0.881 (95% CI: 0.853-0.909). If using the threshold value of 0.234, the sensitivity and specificity of the TBAg/PHA ratio in distinguishing ATB from LTBI were 69.5% (239/344) and 94.12% (192/204). The validation data showed that the performance of the TBAg/PHA ratio in distinguishing ATB from LTBI was also satisfactory, and the area under the ROC curve was 0.901 (95% CI: 0.872-0.931). Furthermore, the TBAg/PHA ratio had an important role in the diagnosis of extra-pulmonary tuberculosis. If using the threshold value of 0.234, the sensitivity and specificity of the TBAg/PHA ratio in the diagnosis of extra-pulmonary tuberculosis were 79.2% (76/96) and 94.1% (192/204). The area under the ROC curve was 0.932 (95% CI: 0.897-0.967). Conclusions: The TBAg/PHA ratio in T-SPOT assay was better than directly using T-SPOT results in distinguishing ATB from LTBI. This ratio also showed a potential use in the diagnosis of extra-pulmonary tuberculosis.

Projections from the dorsomedial division of the bed nucleus of the stria terminalis to hypothalamic nuclei in the mouse.

As stressful environment is a potent modulator of feeding, we seek in the present work to decipher the neuroanatomical basis for an interplay between stress and feeding behaviors. For this, we combined anterograde and retrograde tracing with immunohistochemical approaches to investigate the patterns of projections between the dorsomedial division of the bed nucleus of the stria terminalis (BNST), well connected to the amygdala, and hypothalamic structures such as the paraventricular (PVH) and dorsomedial (DMH), the arcuate (ARH) nuclei and the lateral hypothalamic areas (LHA) known to control feeding and motivated behaviors. We particularly focused our study on afferences to proopiomelanocortin (POMC), agouti-related peptide (AgRP), melanin-concentrating-hormone (MCH) and orexin (ORX) neurons characteristics of the ARH and the LHA, respectively. We found light to intense innervation of all these hypothalamic nuclei. We particularly showed an innervation of POMC, AgRP, MCH and ORX neurons by the dorsomedial and dorsolateral divisions of the BNST. Therefore, these results lay the foundation for a better understanding of the neuroanatomical basis of the stress-related feeding behaviors.

Nutritional and anti-nutritional potential of three accessions of itching bean (Mucuna pruriens (L.) DC var. pruriens): an under-utilized tribal pulse.

Three accessions of the under-utilized legume itching bean (Mucuna pruriens var. pruriens) were analysed for proximate composition, mineral profiles, vitamins (niacin and ascorbic acid), fatty acid profiles, amino acid profiles of total seed protein, in vitro protein digestibility and certain anti-nutritional factors. All three accessions of M. pruriens var. pruriens contained higher amounts of crude protein and crude lipid when compared with most of the commonly consumed pulses. The fatty acid profiles revealed that the seed lipids contained a higher concentration of palmitic acid and linoleic acids. Amino acid profiles of M. pruriens var. pruriens revealed that the seed protein contained relatively higher levels of certain essential amino acids compared with the FAO/WHO requirement pattern. The investigated seeds are rich in minerals such as potassium, calcium, magnesium, phosphorus, iron and manganese. Anti nutritional substances such as total free phenolics, tannins, 3,4-dihydroxyphenylalanine, phytic acid, hydrogen cyanide, trypsin inhibitor activity, oligosaccharides and phytohaemagglutinating activity were investigated. The anti-nutritional fatty acid, behenic acid, also was detected in the present study.

The Use of TB-Specific Antigen/Phytohemagglutinin Ratio for Diagnosis and Treatment Monitoring of Extrapulmonary Tuberculosis.

Extrapulmonary tuberculosis (EPTB) has become more common in recent years; however, the diagnosis of EPTB remains a challenge. In this study, we analyzed the performance of the ratio of TB-specific antigen (TBAg) to phytohemagglutinin (PHA) (TBAg/PHA ratio) in T-SPOT.TB (T-SPOT) assay for diagnosis and treatment monitoring of EPTB. Between 2012 and 2017, 734 EPTB patients were diagnosed and recruited from Tongji hospital, and 1,137 suspected EPTB patients who had other diagnoses were recruited as non-EPTB controls. To validate the study, another small group of EPTB patients and non-EPTB controls were recruited from Sino-French New City Branch of Tongji Hospital. The positive rate of peripheral blood T-SPOT in EPTB and non-EPTB were 88.15 and 32.28%. In T-SPOT positive patients, the direct T-SPOT results had limited value in distinguishing these two conditions. A further calculation of the TBAg/PHA ratio of T-SPOT showed improved performance in each form of EPTB. If using 0.20 as the threshold value of the TBAg/PHA ratio, the pooled sensitivity and specificity were 70.79 and 91.55% in distinguishing EPTB from non-EPTB. The validation results showed a better performance of the TBAg/PHA ratio in distinguishing these two conditions, with a sensitivity and specificity of 81.82 and 97.56%, respectively. Comparing with directly using T-SPOT results, the TBAg/PHA ratio was less affected by immunosuppression. Furthermore, PHA value reflected immunosuppression and could help to judge the credibility of T-SPOT results in EPTB patients with different immune status. The TBAg/PHA ratio was significantly decreased during anti-tuberculosis (TB) treatment, which suggests that it can also be used to monitor therapeutic efficacy. These data provide new insights into the role of T-SPOT assay in TB disease, and the TBAg/PHA ratio might be a useful tool for diagnosis and treatment monitoring of EPTB.

Diagnostic Performance of a 5-Marker Predictive Model for Differential Diagnosis Between Intestinal Tuberculosis and Crohn's Disease.

Background: The differentiation between intestinal tuberculosis (ITB) and Crohn's disease (CD) is a challenge. The aim of this study was to investigate a predictive model for differential diagnosis between ITB and CD. Methods: A total of 268 patients who were suspected of having ITB or CD were prospectively recruited between January 2013 and September 2016. The clinical, laboratory, radiological, endoscopic, and histological features were investigated and subjected to univariate and multivariate analyses. The final predictive model was developed based on the regression coefficients of multivariate logistic regression. To validate the model, the same regression equation was tested on the other group. Results: A total of 239 patients had a final diagnosis, including 86 ITB and 153 CD. Five variables (perianal disease, pulmonary involvement, longitudinal ulcer, left colon, and ratio of tuberculosis-specific antigen to phytohaemagglutinin) were selected for the predictive model to discriminate between ITB and CD. In the predictive model of the training data set, the area under the receiver operating characteristic (ROC) curve, sensitivity, specificity, and accuracy, with a cutoff level of 0.29, were 0.975 (95% confidence interval [CI], 0.939-0.993), 96.7%, 90.7%, and 92.8%, respectively. Application of the predictive model to the validation data set showed similar performance in distinguishing ITB from CD. The area under the ROC curve, sensitivity, specificity, and accuracy were 0.950 (95% CI, 0.871-0.987), 88.5%, 93.5%, and 91.7%, respectively. Conclusions: This 5-marker predictive model could be conveniently used by clinicians to draw a reliable differential diagnosis between ITB and CD in clinical practice. 10.1093/ibd/izy154_video1izy154.video15790725497001.

Use of TBAg/PHA ratio in distinguishing tuberculoma from cancer in solitary pulmonary nodule or mass.

INTRODUCTION: Differentiation of tuberculoma from cancer in solitary pulmonary nodule or mass still remains a major challenge in diagnostic laboratories. OBJECTIVES: The objective of this study is to determine the performance of T-SPOT.TB assay in discriminating these 2 diseases. METHODS: We prospectively enrolled 331 patients with a solitary pulmonary nodule or mass on computed tomography scans. Conventional tests and T-SPOT.TB assay were simultaneously performed in all participants. RESULTS: Our results showed that the performance of directly using T-SPOT.TB results in distinguishing tuberculoma from cancer in solitary pulmonary nodule or mass was not satisfactory because of moderate sensitivity and specificity. However, a further calculation of the ratio of TB-specific antigen (TBAg) to phytohemagglutinin (PHA) (TBAg/PHA ratio) of T-SPOT.TB assay may lead to improvement in distinguishing these 2 diseases. If using the threshold value of 0.236, the sensitivity and specificity of the TBAg/PHA ratio in distinguishing tuberculoma from cancer in solitary pulmonary nodule or mass were, respectively, 80.6% and 93.3%. The area under the curve (AUC) of the receiver operating characteristic curve was 0.921 (95% confidence interval, 0.875-0.967). Furthermore, the TBAg/PHA ratio may also be used to distinguish tuberculoma from other benign diseases (AUC: 0.909, sensitivity: 85.07%, specificity: 90%). CONCLUSIONS: Calculation of the TBAg/PHA ratio might provide a useful non-invasive tool for distinguishing tuberculoma from cancer in patients with a solitary pulmonary nodule or mass in TB-endemic countries.

The performance of the TBAg/PHA ratio in the diagnosis of active TB disease in immunocompromised patients.

OBJECTIVES: The results of the T-SPOT.TB (T-SPOT) assay are reduced in immunocompromised patients with active tuberculosis (ATB), and it is difficult using T-SPOT results to distinguish ATB from latent tuberculosis infection (LTBI) in this condition. The aim of this study was to determine the performance of the TBAg/PHA ratio in T-SPOT assay in the diagnosis of ATB in immunocompromised patients. METHODS: One hundred and forty three immunocompromised ATB patients and 124 LTBI individuals were diagnosed according to conventional tests and T-SPOT assay. RESULTS: The results of T-SPOT assay are of no value in the diagnosis of ATB in immunocompromised patients. However, the number of phytohaemagglutinin (PHA) spot-forming cells (sfc) in T-SPOT assay was substantially decreased in immunocompromised ATB patients compared with that in LTBI individuals. Receiver operating characteristic (ROC) analysis revealed that a further calculation of the TBAg/PHA ratio (the larger of the ESAT-6/PHA and CFP-10/PHA) showed a better performance in distinguishing these two diseases. Using the threshold value of 0.316, the sensitivity and specificity for distinguishing immunocompromised ATB patients from LTBI individuals were respectively 79.21 and 94.05%. CONCLUSIONS: Our findings suggest that the TBAg/PHA ratio might have some significance for the diagnosis of TB disease in immunocompromised patients.

Analysis of unconventional approaches for the rapid detection of surface lectin binding ligands on human cell lines.

For over a decade our laboratory has developed and used a novel histochemical assay using derivatized agarose beads to examine the surface properties of various cell types. Most recently, we have used this assay to examine lectin binding ligands on two human cell types, CCL-220, a colon cancer cell line, and CRL-1459, a non-cancer colon cell line. We found that CCL-220 cells bound specific lectins better than CRL-1459, and this information was used to test for possible differential toxicity of these lectins in culture, as a possible approach in the design of more specific anti-cancer drugs. Although we have examined the validity of the bead-binding assay in sea urchin cell systems, we have not previously validated this technique for mammalian cells. Here the binding results of the bead assay are compared with conventional fluorescence assays, using lectins from three species (Triticum vulgaris, Phaseolus vulgaris, and Lens culinaris) on the two colon cell lines. These lectins were chosen because they seemed to interact with the two cell lines differently. Binding results obtained using both assays were compared for frozen, thawed and fixed; cultured and fixed; and live cells. Both qualitative and quantitative fluorescence results generally correlated with those using the bead assay. Similar results were also obtained with all of the three different cell preparation protocols. The fluorescence assay was able to detect lower lectin binding ligand levels than the bead assay, while the bead assay, because it can so rapidly detect cells with large numbers of lectin binding ligands, is ideal for initial screening studies that seek to identify cells that are rich in surface binders for specific molecules. The direct use of frozen, thawed and fixed cells allows rapid mass screening for surface molecules, without the requirement for costly and time consuming cell culture.

Using the TBAg/PHA ratio in the T-SPOT(®).TB assay to distinguish TB disease from LTBI in an endemic area.

SETTING: An important limitation of the T-SPOT(®).TB assay is its inability to distinguish active tuberculosis (TB) from latent tuberculous infection (LTBI). OBJECTIVE: We proposed a new calculation method for the T-SPOT assay and assessed its effect on distinguishing active TB from LTBI. DESIGN: A total of 162 active TB patients and 97 LTBI individuals were diagnosed according to conventional tests and the T-SPOT assay. RESULTS: The results of early secreted antigenic target 6 (ESAT-6) and culture filtrate protein 10 (CFP-10) in T-SPOT cannot be recommended for distinguishing TB from LTBI. The number of phytohaemagglutinin (PHA) spot-forming cells (sfc) in the T-SPOT assay was reduced in active TB patients. The ESAT-6/PHA or CFP-10/PHA ratios in active TB patients were significantly higher than in individuals with LTBI. Using 0.295 as the threshold ratio of Mycobacterium tuberculosis-specific antigen (TBAg) sfc to PHA sfc (TBAg/PHA ratio, the larger of ESAT-6/PHA and CFP-10/PHA), the sensitivity and specificity were 82.1% and 90.7% in distinguishing active TB from LTBI. The TBAg/PHA ratio might also be used to monitor the effect of anti-tuberculosis treatment. CONCLUSIONS: Calculating the TBAg/PHA ratio might have the potential to diagnose active TB and distinguish TB disease from LTBI.

Purification and characterization of a glycopeptide derived from Phaseolus vulgaris leukoagglutinating phytohemagglutinin.

A glycopeptide obtained from tryptic digestion of citraconylated leukoagglutinating (L-subunit) phytohemagglutinin was isolated and purified. The composition of its ten amino acid and seven hexose residues, when compared to a previous analysis of the NH2-terminal amino acid sequence, indicated that the glycopeptide was derived from residues 11 through 20, with the oligosaccharide unit linked N-glycosidically to asparagine at the twelfth position. Structural studies involving a combination of alpha-mannosidase digestion, periodate oxidation, and permethylation, in conjunction with computerized mass-spectrometric and masschromatographic techniques, suggested a branched sequence of five mannose and two glucosamine residues similar to that found in many mammalian glycoproteins.

Expression of N-acetylglucosaminyltransferase v is associated with prognosis and histology in non-small cell lung cancers.

PURPOSE: N-Acetylglucosaminyltransferase V (GnT-V), a key enzyme in the formation of branching of asparagine-linked oligosaccharides, is strongly linked to tumor invasion and metastasis of colon and breast cancers. However, GnT-V is expressed in many tissues, including normal lung. GnT-V expression has not been examined previously in human lung cancers. The objective of this study is to examine GnT-V expression in non-small cell lung cancers (NSCLCs) and to determine its relationship to biological and clinicopathological characteristics and prognosis. EXPERIMENTAL DESIGN: GnT-V expression was studied by immunohistochemistry in 217 surgically resected NSCLCs and analyzed statistically in relation to various characteristics. RESULTS: High GnT-V expression was found in 113 (52.1%) NSCLCs, and low GnT-V expression was found in 104 (47.9%) NSCLCs. Multivariate logistic regression analysis revealed a significant association between low GnT-V expression and squamous cell carcinomas, as compared with nonsquamous cell carcinomas (P = 0.02). Among biological characteristics of tumors, Ki-67 labeling index was higher in tumors with low GnT-V expression than in those with high GnT-V expression, although this difference was not statistically significant (P = 0.09). Patients with tumors having low GnT-V expression had significantly shorter survival time than patients with tumors having high GnT-V expression in 103 patients with pStage I NSCLCs (5-year survival rates, 49% and 86%, respectively; P = 0.0009), as well as in 59 patients with pStage I non-squamous cell carcinomas (5-year survival rates, 54% and 89%, respectively; P = 0.007). Low GnT-V expression was a significant unfavorable prognostic factor in pStage I NSCLCs (hazard ratio, 2.86; P = 0.002) and in pStage I nonsquamous cell carcinomas (hazard ratio, 3.02; P = 0.02). Furthermore, beta1-6 branching of asparagine-linked oligosaccharides, which are products of GnT-V, were increased highly or moderately in 8 of 10 tumors with high GnT-V expression, as judged by leukoagglutinating phytohemagglutinin staining. CONCLUSIONS: GnT-V expression is associated with histology in NSCLCs. Low GnT-V expression is associated with shorter survival and poor prognosis in pStage I overall NSCLCs and non-squamous cell carcinomas.

Immobilized zinc affinity chromatography of pectin hydroxamic acids for purification of trypsin inhibitors from soybean and sweet potato.

Commercial pectin (with a 94% degree of esterification, DE94) suspended in methanol was reacted with methanolic alkaline hydroxylamine at room temperature for 20 h to prepare pectin hydroxamic acids (PHAs). The prepared PHA was coupled to the epoxy-activated Sepharose 6B gel to get immobilized PHA resins. The immobilized PHA resin was then balanced in column with 2 mM ZnCl2 in 50 mM Tris-HCl buffer (pH 7.9) to test the immobilized Zn-PHA gel as solid phase for immobilized metal affinity chromatography for the purification of trypsin inhibitors (TIs) from soybean and sweet potato. Using TI activity staining, it was found that purified TIs from the commercial soybean and sweet potato after trypsin affinity column purification could be adsorbed onto an immobilized Zn-PHA affinity column and eluted by 100 mM EDTA in 10 mM Tris-HCl buffer (pH 7.9). The immobilized Zn-PHA affinity column was used for TI purifications from crude extracts of sweet potato. The recovery of TI activity for one step was 90%, with 19.74-fold purification increase.

Acute lymphoblastic leukemia blast cells do not inhibit bone marrow hematopoietic progenitor colony formation.

In newly diagnosed patients with acute lymphoblastic leukemia (ALL), bone marrow (BM) morphology always shows "replacement" by blasts with decreased or absent normal hematopoietic elements. To answer the question of whether ALL blasts inhibit replication and maturation of normal marrow progenitors, we studied the interaction of normal marrow with BM specimens from 16 new cases of ALL. Irradiated ALL blasts, supernatant derived from ALL blasts in suspension cultures, and conditioned medium prepared from ALL blasts augmented the colony-forming ability of normal marrow erythroid burst-forming unit (BFU-E), granulocyte-macrophage colony-forming unit (CFU-GM), megakaryocyte colony-forming unit (CFU-MK), and multilineage colony-forming unit (CFU-GEMM) progenitors. In sharp contrast to published data on the suppressive effects of acute myeloblastic leukemia cells on normal hematopoiesis in vitro, our results indicate that the growth advantage of ALL cells over normal marrow elements is not mediated through an inhibitory mechanism derived from leukemia cells.

An ultrasensitive impedimetric glycan biosensor with controlled glycan density for detection of lectins and influenza hemagglutinins.

An impedimetric glycan biosensor with optimised glycan density was applied for the detection of lectins and influenza hemagglutinins down to attomolar concentrations (aM).

Organization of parabrachial projections from the spinal trigeminal nucleus oralis: an anterograde tracing study in the rat.

In recent years, we have accumulated data showing that the spinal trigeminal nucleus oralis (Sp5O) contributes to the processing of somatosensory inputs from the orofacial region. Although the parabrachial area (PB) represents the main brainstem relay for autonomic, nociceptive, and gustatory afferents, few data are available regarding the topographical distribution of the efferent projections from the Sp5O to the PB. We have addressed this question with the rat, by using the anterograde tracer Phaseolus vulgaris leucoagglutinin. A dense trigeminoparabrachial pathway from the Sp5O toward, predominantly, the ipsilateral PB was revealed. Projections come mainly from the dorsal part of the Sp5O that was found to innervate densely the medial, external medial, and ventral lateral subnuclei. In contrast, the ventral part of the Sp5O projected almost exclusively to an as yet not formally described region, located dorsally and laterally to the lateral tip of the brachium conjunctivum, close to the Kölliker-Fuse nucleus. These results suggest that distinct regions within the Sp5O may be involved in the processing of gustatory and nociceptive information.

Supracapsular bed nucleus of the stria terminalis contains central and medial extended amygdala elements: evidence from anterograde and retrograde tracing experiments in the rat.

Neurons that accompany the stria terminalis as it loops over the internal capsule have been termed collectively the supracapsular bed nucleus of the stria terminalis (BSTS). They form two cell columns, a lateral column and a considerably smaller medial column. The lateral column merges rostrally with the lateral bed nucleus of the stria terminalis and caudally with the central amygdaloid nucleus (central extended amygdala components). The medial column is continuous with the medial bed nucleus of the stria terminalis and the medial amygdaloid nucleus (medial extended amygdala districts). The connections of the BSTS were investigated in the rat by placing injections of Phaseolus vulgaris-leucoagglutinin (PHA-L) or retrograde tracers in different parts of the extended amygdala or in structures related to the extended amygdala. BSTS inputs and outputs were identified, respectively, by the presence of varicose fibers and retrogradely labeled neurons within the stria terminalis. The results suggest that the medial-to-lateral compartmentalization of BSTS neurons reflects their close alliance with the medial and central divisions of the extended amygdala. The medial BSTS contains primarily elements that correspond to the posterodorsal part of the medial amygdaloid nucleus and the medial column of the posterior division of the medial bed nucleus of the stria terminalis, and the lateral BSTS contains elements that correspond to the medial and lateral parts of the central amygdaloid nucleus and lateral bed nucleus of the stria terminalis. These results add strong support to the concept of the extended amygdala as a ring-like macrostructure around the internal capsule, and they are of theoretical interest for the understanding of the organization of the basal forebrain.

The striatum and the globus pallidus send convergent synaptic inputs onto single cells in the entopeduncular nucleus of the rat: a double anterograde labelling study combined with postembedding immunocytochemistry for GABA.

The entopeduncular nucleus is one of the major output stations of the basal ganglia. In order to better understand the role of this structure in information flow through the basal ganglia, experiments have been performed in the rat to examine the chemical nature, morphology, and synaptology of the projections from the globus pallidus and striatum to the entopeduncular nucleus. In order to examine the morphology and synaptology of pallidoentopeduncular terminals and striatoentopeduncular terminals, rats were subjected to a double anterograde labelling study. The globus pallidus was injected with Phaseolus vulgaris-leucoagglutinin (PHA-L), and on the same side of the brain, the striatum was injected with biocytin. The entopeduncular nuclei of these animals were then examined for anterogradely labelled pallidal and striatal terminals. Rich plexuses of PHA-L-labelled pallidal terminals and biocytin-labelled striatal terminals were identified throughout the entopeduncular nucleus. At the electron microscopic level, the pallidal boutons were classified as two types. The majority (Type 1), were large boutons that formed symmetrical synapses with the dendrites and perikarya of neurones in the entopeduncular nucleus. Type 2 PHA-L-labelled terminals were much rarer, slightly smaller, and formed asymmetrical synapses. It is suggested that the Type 2 boutons are not derived from the globus pallidus but from the subthalamic nucleus. The biocytin-labelled terminals from the striatum had the typical morphological features of striatal terminals and formed symmetrical synapses. The distribution of the postsynaptic targets of the pallidal terminals and the striatal terminals differed in that the pallidal terminals preferentially made synaptic contact with the more proximal regions of the neurones in the entopeduncular nucleus, whereas the striatal terminals were located more distally on the dendritic trees. Examination in the electron microscope of areas where there was an overlap of the two sets of anterogradely labelled boutons revealed that terminals from the globus pallidus and the striatum made convergent synaptic contact with the perikarya and dendrites of individual neurones in the entopeduncular nucleus. In order to examine the chemical nature of the input to the entopeduncular nucleus from the globus pallidus and the striatum, ultrathin sections were immunostained by the postembedding method to reveal endogenous GABA. Three classes of GABA-containing terminals were identified; two of them formed symmetrical synapses and one rare type formed asymmetrical synapses. The combination of the GABA immunocytochemistry and anterograde labelling revealed that both the striatal and pallidal afferents that make symmetrical synapses with neurones in the entopeduncular nucleus, including those involved in convergent inputs, are GABAergic.(ABSTRACT TRUNCATED AT 400 WORDS)

Ascending projections from the area around the spinal cord central canal: A Phaseolus vulgaris leucoagglutinin study in rats.

A single small iontophoretic injection of Phaseolus vulgaris leucoagglutinin labels projections from the area surrounding the spinal cord central canal at midthoracic (T6-T9) or lumbosacral (L6-S1) segments of the spinal cord. The projections from the midthoracic or lumbosacral level of the medial spinal cord are found: 1) ascending ipsilaterally in the dorsal column near the dorsal intermediate septum or the midline of the gracile fasciculus, respectively; 2) terminating primarily in the dorsal, lateral rim of the gracile nucleus and the medial rim of the cuneate nucleus or the dorsomedial rim of the gracile nucleus, respectively; and 3) ascending bilaterally with slight contralateral predominance in the ventrolateral quadrant of the spinal cord and terminating in the ventral and medial medullary reticular formation. Other less dense projections are to the pons, midbrain, thalamus, hypothalamus, and other forebrain structures. Projections arising from the lumbosacral level are also found in Barrington's nucleus. The results of the present study support previous retrograde tract tracing and physiological studies from our group demonstrating that the neurons in the area adjacent to the central canal of the midthoracic or lumbosacral level of the spinal cord send long ascending projections to the dorsal column nucleus that are important in the transmission of second-order afferent information for visceral nociception. Thus, the axonal projections through both the dorsal and the ventrolateral white matter from the CC region terminate in many regions of the brain providing spinal input for sensory integration, autonomic regulation, motor and emotional responses, and limbic activation.

Blotting methods applied to investigations of the mitogenic activity of phytohaemagglutinin (PHA).

PHA polypeptides were separated by SDS-polyacrylamide gel electrophoresis and transferred to nitrocellulose filters which were then cut into identical discs and used to perform blastogenic stimulation on cultured human peripheral blood lymphocytes. This method offers the following advantages: the high resolution of SDS-polyacrylamide gel electrophoresis, the sensitive immunochemical identification of the polypeptides involved in the mitogenic responses of lymphocytes, the determination in the same experiment of the molecular weight, antigenicity and biological activities of the proteins.

The relationship between metal ion content and mitogenicity of PHA-L4 isolectin as determined by agarose electrophoresis.

The lectin (PHA) from Phaseolus vulgaris is a tetrameric metalloprotein which exists as 5 different isolectins. An agarose electrophoretic assay is described that can distinguish between metallized and demetallized PHA. The assay has been employed for the analysis of 5 different preparations of the isolectin PHA-L4, and the preparations have subsequently been tested for their ability to induce lymphocyte transformation. A reverse correlation was found between the relative amount of irreversibly demetallized isolectin, as determined by agarose electrophoresis and the mitogenic activity of the preparation. The degree of irreversible demetallization seemed to be dependent on the preparation method used. One method gave 25% and another 100% demetallization of the isolectins. The assay was found to be a rapid and simple way to test the relative amount of mitogenically active lectin present in a preparation.